The biogenesis of chloroplast organelle has been described in great detail. However, description of the transcriptional activity of this DNA is missing and very little is known about the transcriptional regulation of organelle gene expression. The proposed research will concentrate on the determination of spacial and temporal regulation of organelle gene expression. Using synchronously grown cultures, and the mutant Y1 of C. reinhardi, mRNA synthesis in the chloroplast will be investigated; and with the use of Ch1DNA fragments generated by restriction enzymes, the position of the mRNA's on the genome will be mapped. The in vivo synthesized mRNA will be compared to that obtained in vitro in an attempt to determine the nature and the sites of chloroplast promoters on DNA. The RNA polymerase holoenzyme of E. coli forms sigma-dependent, preinitiation complexes at 18 to 38 specific sites on chloroplast DNA of C. reinhardi. These complexes are functionally indistinguishable from complexes formed by this enzyme at promoter sites on DNA of coliphages (Surzycki et al., 1976). Moreover, chloroplast RNA polymerase and its initiation factor showed a great similarity to those of E. coli (Ratcliff & Surzycki 1974, Surzycki & Shellenberger 1976). We will investigate the relationship between initiation sites for E. coli and chloroplast RNA polymerases on chloroplast DNA. First, the specificity of initiation sites can be compared by competition for binding sites by different RNA polymerases. Second transcriptional complexes can be stratified and mapped $ electron microscopically using fragments of chloroplast DNA derived from restriction enzymes. Third, the relation of RNA made in vitro to that observed in vivo will be studied by competition hybridization. Hopefully, our investigation will provide insights into the temporal and spacial control of gene expression during biogenesis of organelles. In addition, we should obtain information pertaining to the mechanism of initiation of transcription by RNA polymerases in eukaryotic cells and the role of initiation factors in this process.